Ad5/35 mCD20载体的构建及鉴定
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摘要
【目的】B细胞来源的淋巴瘤是淋巴瘤中最常见的一种类型,而免疫治疗在该类型淋巴瘤的治疗中具有重要的作用;编码肿瘤相关抗原腺病毒载体感染树突状细胞(Dendritic cells,DC)是一种很有前景的肿瘤疫苗。然而,目前常用的5型腺病毒载体感染DC的效率较低。本研究的目的在于比较5型和5/35型腺病毒载体感染DC的效率,并在此基础上克隆小鼠CD20(mCD20)基因、构建编码mCD20的5/35型腺病毒载体(Ad5/35 mCD20),并鉴定其表达产物,为编码CD20基因的5/35型腺病毒载体修饰DC作为一种B细胞淋巴瘤的免疫基因治疗手段提供实验依据和前期的基础。
【方法】从健康人外周血单个核细胞中培养DC,用不同病毒滴度的Ad5 EGFP或Ad5/35 EGFP感染DC,用流式细胞仪双染色,检测EGFP的表达,比较两型不同的腺病毒载体感染DC的效率。运用分子克隆技术,从C57BL/6小鼠中克隆小鼠CD20,经PCR、酶切进行鉴定并测序正确后,将该基因cDNA片段插入腺病毒穿梭质粒pDC315中,构建pDC315 mCD20。采用Ad Max~(TM)腺病毒载体包装体系,将pDC315mCD20与含35型腺病毒纤维的腺病毒骨架质粒通过磷酸钙沉淀法共转染至293细胞中,经反复扩增、纯化获得Ad5/35 mCD20。将鉴定后的Ad5/35mCD20转染至HepG2细胞中,经抗鼠CD20单克隆抗体染色,通过流式细胞技术检测CD20的表达。
【结果】用每个细胞100、50和10 pfu的Ad5 EGFP或Ad5/35 EGFP感染人DC,经流式细胞仪CD11a-PE和EGFP双染色,其感染效率分别为28.41%,17.36%,3.22%and69.12%,49.03%,36.89%。从C57BL/6小鼠克隆的mCD20 cDNA经测序证实与GeneBank提供的序列(No.M62541)完全一致。Ad5/35mCD20转染HepG2细胞24h,流式细胞仪检测其表达为38.72%。
【结论】两型腺病毒载体感染DC的效率与病毒感染的滴度成正比,滴度愈大其感染DC的效率愈高;在相同的病毒滴度时,5/35型腺病毒载体感染DC的效率明显地高于5型腺病毒载体。将小鼠CD20基因插入E1区缺失的5/35型腺病毒载体构建的Ad5/35 mCD20能有效地表小鼠CD20蛋白,为后续研究编码CD20基因的5/35型腺病毒载体修饰DC作为B细胞淋巴瘤免疫基因治疗的一种手段提供一种有用的工具。
[Objective]: B-cell lymphoma is one of the most common types of lymphomas,and immunotherapy plays an important role in the treatment of the lymphoma. Dendritic cells (DC) which is infected by adenovirus and encode as the tumor associated antigen (TAA) is a promising neoplasm tumor vaccine, however, the commonly used adenovirus subgenus C serotypes 5 (Ad5) carrier DC infections is of low efficiency. This research aims at comparing the efficiency of adenovirus subgenus C serotypes 5(Ad5) carrier DC infection with adenovirus subgenus B serotypes 5/35 (Ad5/35) carrier DC infection, cloning the CD20 (mCD20) gene of mouse, establishing the adenovirus vector 5/35 of coding mCD20 (Ad5/35mCD20), identifying expression outcomes,providing experimental evidence and initial basis for DC modification of adenovirus vector 5/35 coding CD20 gene being used as the immune gene therapy of B-cell lymphoma.
[Method]: To culture DC in Peripheral blood mononuclear cell (PBMC) in the healthy people, infect DC with Ad5EGFP or Ad5/35EGFP of different virus tites, double-dye it with the flow cytometry device,check the expression of EGFP,and then compare the efficiency of the two different adenovirus vector infecting DC. The molecular clonal technology is employed to clone the mouse CD20 from mouse C57BL/6, insert cDNA segment of the gene into adenovirus shuttle plasmid pDC315 and establish pDC315mCD20 after PCR, restriction enzyme digesting, identification and DNA sequencing being confirmed. Ad MaxTM adenovirus vector packing system is employed to transfect pDC315mCD20 and the adenovirus framework plasmid containing adenovirus vector 5/35 fiber to 293 cells through the calcium phosphate precipitation method, and obtain Ad5/35mCD20 by repeated amplification and purification. The plasmid encodging the fusion genemCD20 was transfected into HepG2 cells which will be detected and identified by flow cytometry.
[Result]: To infect the human DC with 100,50 or 10 pfu Ad5EGFP and Ad5/35EGFP of each cell, after double-dying CD11 a-PE and EGFP through the flow cytometry device the infection efficiencies are 28.41%,17.36%, 3.22% and 69.12%,49.03%,36.89% respectively. The cloned mCD20cDNA from the mouse C57BL/6 proves through DNA sequencing to have the same sequence (No.M62541) provided by Gene Bank. The plasmid was transfected into HepG2 cells and the stable expression clone was obtained. After dying CD20, the expression of mCD20 in the cells which is 38.72% was detected by flow cvtometry.
[Conclusion]: The efficiencies of the two adenovirus vector carrier DC infections are in direct ratio with the tites of virus infection, and the higher tite will have the higher efficiency of DC infection; with the same virus tite, 5/35 adenovirus vector has an obviously higher efficiency of DC infection than adenovirus subgenus C serotypes 5 (Ad5). The Ad5/35mCD20 established by inserting the gene of the mouse CD20 into 5/35 adenovirus vector without section El can effectively express protein of the mouse CD20, thereby providing a useful tool for further research on 5/35 adenovirus vector coding CD20 gene, DC modification could be used as an approach for the immune gene therapy of B-cell lymphoma.
【方法】从健康人外周血单个核细胞中培养DC,用不同病毒滴度的Ad5 EGFP或Ad5/35 EGFP感染DC,用流式细胞仪双染色,检测EGFP的表达,比较两型不同的腺病毒载体感染DC的效率。运用分子克隆技术,从C57BL/6小鼠中克隆小鼠CD20,经PCR、酶切进行鉴定并测序正确后,将该基因cDNA片段插入腺病毒穿梭质粒pDC315中,构建pDC315 mCD20。采用Ad Max~(TM)腺病毒载体包装体系,将pDC315mCD20与含35型腺病毒纤维的腺病毒骨架质粒通过磷酸钙沉淀法共转染至293细胞中,经反复扩增、纯化获得Ad5/35 mCD20。将鉴定后的Ad5/35mCD20转染至HepG2细胞中,经抗鼠CD20单克隆抗体染色,通过流式细胞技术检测CD20的表达。
【结果】用每个细胞100、50和10 pfu的Ad5 EGFP或Ad5/35 EGFP感染人DC,经流式细胞仪CD11a-PE和EGFP双染色,其感染效率分别为28.41%,17.36%,3.22%and69.12%,49.03%,36.89%。从C57BL/6小鼠克隆的mCD20 cDNA经测序证实与GeneBank提供的序列(No.M62541)完全一致。Ad5/35mCD20转染HepG2细胞24h,流式细胞仪检测其表达为38.72%。
【结论】两型腺病毒载体感染DC的效率与病毒感染的滴度成正比,滴度愈大其感染DC的效率愈高;在相同的病毒滴度时,5/35型腺病毒载体感染DC的效率明显地高于5型腺病毒载体。将小鼠CD20基因插入E1区缺失的5/35型腺病毒载体构建的Ad5/35 mCD20能有效地表小鼠CD20蛋白,为后续研究编码CD20基因的5/35型腺病毒载体修饰DC作为B细胞淋巴瘤免疫基因治疗的一种手段提供一种有用的工具。
[Objective]: B-cell lymphoma is one of the most common types of lymphomas,and immunotherapy plays an important role in the treatment of the lymphoma. Dendritic cells (DC) which is infected by adenovirus and encode as the tumor associated antigen (TAA) is a promising neoplasm tumor vaccine, however, the commonly used adenovirus subgenus C serotypes 5 (Ad5) carrier DC infections is of low efficiency. This research aims at comparing the efficiency of adenovirus subgenus C serotypes 5(Ad5) carrier DC infection with adenovirus subgenus B serotypes 5/35 (Ad5/35) carrier DC infection, cloning the CD20 (mCD20) gene of mouse, establishing the adenovirus vector 5/35 of coding mCD20 (Ad5/35mCD20), identifying expression outcomes,providing experimental evidence and initial basis for DC modification of adenovirus vector 5/35 coding CD20 gene being used as the immune gene therapy of B-cell lymphoma.
[Method]: To culture DC in Peripheral blood mononuclear cell (PBMC) in the healthy people, infect DC with Ad5EGFP or Ad5/35EGFP of different virus tites, double-dye it with the flow cytometry device,check the expression of EGFP,and then compare the efficiency of the two different adenovirus vector infecting DC. The molecular clonal technology is employed to clone the mouse CD20 from mouse C57BL/6, insert cDNA segment of the gene into adenovirus shuttle plasmid pDC315 and establish pDC315mCD20 after PCR, restriction enzyme digesting, identification and DNA sequencing being confirmed. Ad MaxTM adenovirus vector packing system is employed to transfect pDC315mCD20 and the adenovirus framework plasmid containing adenovirus vector 5/35 fiber to 293 cells through the calcium phosphate precipitation method, and obtain Ad5/35mCD20 by repeated amplification and purification. The plasmid encodging the fusion genemCD20 was transfected into HepG2 cells which will be detected and identified by flow cytometry.
[Result]: To infect the human DC with 100,50 or 10 pfu Ad5EGFP and Ad5/35EGFP of each cell, after double-dying CD11 a-PE and EGFP through the flow cytometry device the infection efficiencies are 28.41%,17.36%, 3.22% and 69.12%,49.03%,36.89% respectively. The cloned mCD20cDNA from the mouse C57BL/6 proves through DNA sequencing to have the same sequence (No.M62541) provided by Gene Bank. The plasmid was transfected into HepG2 cells and the stable expression clone was obtained. After dying CD20, the expression of mCD20 in the cells which is 38.72% was detected by flow cvtometry.
[Conclusion]: The efficiencies of the two adenovirus vector carrier DC infections are in direct ratio with the tites of virus infection, and the higher tite will have the higher efficiency of DC infection; with the same virus tite, 5/35 adenovirus vector has an obviously higher efficiency of DC infection than adenovirus subgenus C serotypes 5 (Ad5). The Ad5/35mCD20 established by inserting the gene of the mouse CD20 into 5/35 adenovirus vector without section El can effectively express protein of the mouse CD20, thereby providing a useful tool for further research on 5/35 adenovirus vector coding CD20 gene, DC modification could be used as an approach for the immune gene therapy of B-cell lymphoma.
引文
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2. Ghielmini M et al. Single agent rituximab in patients with follicular or mantle cell lymphoma: clinical and biological factors that are predictive of response and event-free survival as well as the effect of rituximab on the immune system: a study of the Swiss Group for Clinical Cancer Research (SAKK) Ann Oncol. 2005; 16: 1675-1682.
3. Hiddemann W, et al. Front-Line Therapy with Rituximab added to the Combination of Cyclophosphamide, Doxorubicin, Vincristine and Prednisone (CHOP) significantly improves the Outcome of Patients with Advanced Stage Follicular Lymphomas as compared to CHOP alone - Results of a Prospective Randomized Study of the German Low Grade Lymphoma Study Group (GLSG). Blood, 2005; 106:3725-3732.
4. Mangel J, et al. Pharmacokinetic study of patients with follicular or mantle cell immunotherapy following autologous stem cell transplantation. Ann Oncol. 2003; 14: 758-765.
5. Olivieri A, et al. A New Schedule of CHOP/Rituximab Plus Granulocyte-Macrophage Colony-Stimulating Factor Is an Effective Rescue for Patients with Aggressive Lymphoma Failing Autologous Stem Cell Transplantation. Biol Blood Marrow Transplant, 2005; 11:627-636.
6. Eisenbeis CF, et al. Combination immunotherapy of B-cell non-Hodgkin's lymphoma with rituximab and interleukin-2: a preclinical and phase I study. Clin Cancer Res, 2004; 10:6101-6110.
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